WO2010119307A1 - Method for stimulating antigen-specific t cell responses using accelerated co-cultured dendritic cells - Google Patents

Method for stimulating antigen-specific t cell responses using accelerated co-cultured dendritic cells Download PDF

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WO2010119307A1
WO2010119307A1 PCT/IB2009/052793 IB2009052793W WO2010119307A1 WO 2010119307 A1 WO2010119307 A1 WO 2010119307A1 IB 2009052793 W IB2009052793 W IB 2009052793W WO 2010119307 A1 WO2010119307 A1 WO 2010119307A1
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disease
cell
cells
sample
cell responses
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PCT/IB2009/052793
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English (en)
French (fr)
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Roberto Mallone
Emanuela Martinuzzi
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INSERM (Institut National de la Santé et de la Recherche Médicale)
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Priority to PCT/IB2009/052793 priority Critical patent/WO2010119307A1/en
Priority to EP10714449.5A priority patent/EP2419730B1/en
Priority to US13/264,255 priority patent/US9091679B2/en
Priority to ES10714449.5T priority patent/ES2601105T3/es
Priority to JP2012505147A priority patent/JP5776684B2/ja
Priority to PCT/EP2010/054826 priority patent/WO2010119033A1/en
Publication of WO2010119307A1 publication Critical patent/WO2010119307A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4615—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4648—Bacterial antigens
    • A61K39/464817—Mycobacterium, e.g. Mycobacterium tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4648—Bacterial antigens
    • A61K39/46482—Clostridium, e.g. Clostridium tetani
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the invention relates to a method for stimulating antigen-specific T cell responses.
  • PBMCs peripheral blood mononuclear cells
  • APCs antigen-presenting cells
  • DC circulating dendritic cells
  • IL-2 and IL-7 co-stimulatory cytokines
  • monocytes can first be isolated and differentiated into immature DC with granulocyte/macrophage colony-stimulating factor (GM-CSF) and IL-4 for 5-7 days, to be subsequently matured with different proinflammatory stimuli for an additional 24-48h [Zhou et al, Proc.Natl.Acad.Sci. USA 93:2588, 1996].
  • GM-CSF granulocyte/macrophage colony-stimulating factor
  • IL-4 granulocyte/macrophage colony-stimulating factor
  • This strategy exploits the higher stimulatory potency of DCs to achieve a larger CD4+ T cell expansion. While attractive, it requires however larger starting blood volumes, as monocytes represent only -5-15% of PBMCs, and autologous monocytes should ideally be used to avoid selection of allo-specific CD4+ T cells. T cells need therefore to be kept in culture or frozen down while monocyte-derived DC are being generated. Besides higher PBMC needs,
  • peptide epitopes for T-cell stimulation requires preliminary identification of those epitopes targeted by the immune response. This identification procedure is very labor-intensive, and specific for one HLA Class I or Class II allele. Thus, different epitopes have to be identified for different HLA alleles, depending on the human subjects that one wishes to study.
  • the invention provides a method for stimulating antigen (Ag)-specif ⁇ c T cell responses in a blood sample or PBMC sample isolated from a subject, comprising the following steps: a) culturing said blood or PBMC sample in a medium which induces differentiation of dendritic cells (DC); b) optionally, maturing said DC; wherein an Ag is added during steps a) and/or b).
  • the invention also relates to the use of such a method for diagnosing a disease and/or for monitoring the effects of an immune therapy in a subject.
  • Another aspect of the invention relates to the use of such a method for producing Ag- specific T cell clones. Yet another aspect of the invention relates to the use of such a method for evaluating the immunogenicity of therapeutic proteins and for epitope mapping analyses.
  • the invention relates to a method for stimulating Ag-specif ⁇ c T cell responses in a blood or PBMC sample isolated from a subject, comprising the following steps: a) culturing said blood sample or PBMC sample in a medium which induces the differentiation of DC; b) optionally, maturing said DC; wherein an Ag is added during steps a) and/or b).
  • the Ag-specif ⁇ c T cell responses are CD4+ T cell responses. In another embodiment, the Ag-specif ⁇ c T cell responses are CD8+ T cell responses.
  • suitable biological samples for carrying out the method of the invention are a blood sample or a PBMC sample purified from whole blood using conventional density gradient separation protocols.
  • the biological sample of the invention is a PBMC sample.
  • PBMC peripheral blood mononuclear cells
  • un- fractionated PBMC refers to whole PBMC, i.e. to a population of white blood cells having a round nucleus, which has not been enriched in a given sub-population.
  • the PBMC sample according to the invention has not been subjected to a selection step to contain only adherent PBMC (which consist essentially of >90% monocytes) or non-adherent PBMC (which contain T cells, B cells, natural killer (NK) cells, NK T cells and DC precursors).
  • adherent PBMC which consist essentially of >90% monocytes
  • non-adherent PBMC which contain T cells, B cells, natural killer (NK) cells, NK T cells and DC precursors.
  • a PBMC sample according to the invention therefore contains lymphocytes (B cells, T cells, NK cells, NKT cells), monocytes, and precursors thereof.
  • lymphocytes B cells, T cells, NK cells, NKT cells
  • monocytes and precursors thereof.
  • these cells can be extracted from whole blood using Ficoll, a hydrophilic polysaccharide that separates layers of blood, with the PBMC forming a cell ring under a layer of plasma.
  • PBMC can be extracted from whole blood using a hypotonic lysis which will preferentially lyse red blood cells. Such procedures are known to the expert in the art.
  • the biological sample according to the invention can be a blood sample.
  • blood sample or "unfractionated blood sample” as used herein refers to a crude blood specimen which has been isolated from a subject and collected in tubes or other containers containing an appropriate anti-coagulant (e.g., lithium heparin or sodium citrate).
  • the blood sample is unfractionated whole blood and contains plasma and blood cells (red blood cells, white blood cells). It may be a freshly isolated blood sample ( ⁇ 48h) or a blood sample which has been obtained previously and kept frozen until use.
  • subject refers to a mammalian, such as a rodent (e.g. a mouse or a rat), a feline, a canine or a primate.
  • rodent e.g. a mouse or a rat
  • feline e.g. a feline
  • canine e.g. a canine
  • primate e.g. a primate
  • said subject is a human subject.
  • the subject according to the invention can be a healthy subject or a subject suffering from a given disease.
  • an antigen refers to protein or peptide preparations capable of eliciting a T cell response.
  • said Ag is a protein which can be obtained by recombinant DNA technology or by purification from different tissue or cell sources. Such proteins are not limited to natural ones, but also include modified proteins or chimeric constructs, obtained for example by changing selected amino acid sequences or by fusing portions of different proteins.
  • said Ag is a synthetic peptide, obtained by Fmoc biochemical procedures, large-scale multipin peptide synthesis, recombinant DNA technology or other suitable procedures.
  • the Ag is a crude or partially purified tissue or cell preparation obtained by different biochemical procedures (e.g., lysis, subcellular fractionation, density gradient separation) known to the expert in the art.
  • the method of the invention comprises the step of culturing a blood sample or a PBMC sample in a medium which induces the differentiation of DC.
  • Suitable media for carrying out the invention are any culture medium suitable for growth, survival and differentiation of PBMCs. Typically, it consists of a base medium containing nutrients (a source of carbon, aminoacids), a pH buffer and salts, which can be supplemented with serum of human or other origin and/or growth factors and/or antibiotics, to which agents are added that induce differentiation of DC.
  • the base medium can be RPMI 1640, DMEM, IMDM, X-VIVO or AIM-V medium, all of which are commercially available standard media.
  • RPMI 1640 RPMI 1640, DMEM, IMDM, X-VIVO or AIM-V medium, all of which are commercially available standard media.
  • DMEM DMEM
  • IMDM IMDM
  • X-VIVO X-VIVO
  • AIM-V medium all of which are commercially available standard media.
  • the cell culture may be performed at 37°C in a 5% CO 2 atmosphere, using tissue culture incubators suitable to this end.
  • said medium comprises Granulocyte/Macrophage Colony-Stimulating Factor (GM-CSF).
  • GM-CSF Granulocyte/Macrophage Colony-Stimulating Factor
  • GM-CSF is used in an amount comprised between 1 and 10,000 U/ml, preferably between 10 and 5,000 U/ml, even more preferably at about 1,000 U/ml.
  • GM-CSF can be obtained from a variety of sources. It may be purified or recombinant GM-CSF. GM-CSF is commercially available from different companies, for example R&D Systems or PeproTech. In a preferred embodiment, said medium further comprises interleukin 4 (IL-4). Typically, IL-4 is used in an amount comprised between 0 and 10,000 U/ml, preferably between 10 and 1,000 U/ml, even more preferably at about 500 U/ml. IL-4 can be obtained from a variety of sources. It may be purified or recombinant IL- 4. IL-4 is commercially available from different companies, for example R&D Systems or PeproTech.
  • IL-4 is commercially available from different companies, for example R&D Systems or PeproTech.
  • the step of incubating the blood sample or PBMC sample in a medium which induces the differentiation of DC is performed for an amount of time sufficient for enriching said blood sample or PBMC sample in DC.
  • this can be easily tested by examining the relative expression of markers which are known to be expressed or not to be expressed by DC.
  • an enrichment of the blood sample or PBMC sample may be reflected by an increase in markers such as CDl Ic, HLA-DR, CD80 and CD86 and/or by a decrease in markers such as CD14.
  • the specificity of expression of these markers on DC populations can be assessed by limiting the cells under analysis to selected PBMC or whole blood subsets, using a variety of gating strategies.
  • DC may be identified as cells not expressing markers typical of other subpopulations (e.g., CD3, CD 14, CD 16, CD 19, CD34; so called lin neg cells), and expressing HLA-DR.
  • said step is carried out for an amount of time t(a) comprised between t(a)min and t(a)max.
  • the minimal incubation for step a), t(a)min can be about 12 hours, preferably about 16 hours, even more preferably about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, even more preferably about 24 hours.
  • t(a)max can be about 10 days, preferably about 7 days, even more preferably about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, even more preferably about 1 day.
  • step a) is carried out for an amount of time t(a) comprised between about 16 hours and about 7 days, preferably between about 20 hours and about 4 days.
  • step a) is carried out for an amount of time t(a) of about 24 hours.
  • said DC after enriching the blood sample or PBMC sample in DC during step a), said DC can be matured during step b).
  • pro-inflammatory stimuli and/or agents which mimic a viral or bacterial aggression are added to the medium of step a).
  • pro-inflammatory stimuli suitable for the method of the invention are, but are not limited to, tumor necrosis factor alpha (TNF- ⁇ ), interleukin-1 beta (IL- l ⁇ ), prostaglandin E2 (PGE2), anti-CD40 monoclonal antibodies (mAbs), CD40 ligand (CD40L) recombinant chimeric proteins, interferon-alpha (IFN- ⁇ ), interferon- gamma (IFN- ⁇ ), interleukin-7 (IL-7).
  • TNF- ⁇ tumor necrosis factor alpha
  • IL- l ⁇ interleukin-1 beta
  • PGE2 prostaglandin E2
  • mAbs anti-CD40 monoclonal antibodies
  • CD40L CD40 ligand
  • Such agents can be used alone or in different combinations with other pro-inflammatory stimuli or viral/bacterial mimetic agents.
  • agents which mimic a viral or bacterial aggression suitable for the method of the invention are, but are not limited to, lipopolysaccharides (LPS), CpG oligodeoxynucleotides, polyinosinic:polycytidylic acid (poly I:C), Pam3CysSerLys4 (Pam3CSK4), imiquimod.
  • LPS lipopolysaccharides
  • CpG oligodeoxynucleotides polyinosinic:polycytidylic acid (poly I:C), Pam3CysSerLys4 (Pam3CSK4), imiquimod.
  • Such agents can be used alone or in different combinations with other pro-inflammatory stimuli or viral/bacterial mimetic agents.
  • step b) is carried out in the presence of at least one agent selected from the group consisting of TNF- ⁇ , IL- l ⁇ , PGE2, anti-CD40 antibody,
  • IFN- ⁇ 2a LPS, poly LC, IFN- ⁇ , IL-7 and mixtures thereof.
  • Said agent(s) are agents known to stimulate immune responses, and the skilled person will be able to select the appropriate concentrations of each agent for obtaining DC maturation while limiting non-specific T cell activation.
  • step b) is carried out in the presence of TNF- ⁇ , IL- l ⁇ and PGE2.
  • TNF- ⁇ is used in an amount comprised between 1 and 10,000 U/ml, preferably between 10 and 5,000 U/ml, even more preferably at about 1,000 U/ml.
  • TNF- ⁇ can be obtained from a variety of sources. It may be purified or recombinant
  • TNF- ⁇ is commercially available from different companies, for example
  • IL- l ⁇ is used in an amount comprised between 0.1 and 1,000 ng/ml, preferably between 1 and 100 ng/ml, even more preferably at about 10 ng/ml.
  • IL- l ⁇ can be obtained from a variety of sources. It may be purified or recombinant IL- l ⁇ .
  • IL- l ⁇ is commercially available from different companies, for example R&D
  • PGE2 is used in an amount comprised between 0.01 and 100 ⁇ M, preferably between 0.1 and 10 ⁇ M, even more preferably at about 1 ⁇ M.
  • PGE2 can be obtained from a variety of sources.
  • PGE2 is commercially available as a product of synthesis from different companies, for example Calbiochem/Merck or Sigma.
  • step b) is carried out in the presence of anti-CD40 (for example mAb clone G28-5) and IFN-CC.
  • anti-CD40 mAb is used in an amount comprised between 0.1 and 50 ⁇ g/ml, preferably between 1 and 25 ⁇ g/ml, even more preferably at about 10 ⁇ g/ml.
  • anti-CD40 mAb is clone G28-5. Purified G28-5 or other anti-CD40 mAbs can be produced in-house from hybridoma culture supernatants according to procedures known to those skilled in the art, or purchased from different commercial sources such as BioLegend or eBioscience.
  • anti-CD40 mAbs can be substituted with recombinant CD40 ligand molecules, synthesized either in monomeric or in multimeric form.
  • Recombinant CD40 ligand molecules can be produced in-house using recombinant
  • IFN-CC is used in an amount comprised between 1 and 10,000 U/ml, preferably between 10 and 5,000 U/ml, even more preferably at about 1,000 U/ml.
  • IFN-CC is IFN-Cc2a.
  • IFN-CC can be obtained from a variety of sources. It may be purified or recombinant IFN-CC. IFN-CC is commercially available from different companies, for example Roche (Roferon-A), R&D Systems or
  • step b) is carried out in the presence of LPS.
  • LPS is used in an amount comprised between 1 and 10,000 ng/ml, preferably between 10 and 1,000 ng/ml, even more preferably at about 100 ng/ml.
  • LPS can be obtained from a variety of sources. It may be purified from different bacterial strains. Suitable strains are, but are not limited to, E. coli, K. pneumoniae,
  • LPS is commercially available from different companies, for example Sigma.
  • step b) is carried out in the presence of poly LC.
  • poly LC is used in an amount comprised between 0.1 and 1,000 ⁇ g/ml, preferably between 1 and 100 ⁇ g/ml, even more preferably at about 20 ⁇ g/ml.
  • Poly LC can be obtained from a variety of sources. It may be synthesized using methodologies known to those skilled in the art. Poly I:C is commercially available from different companies, for example Sigma.
  • a low dose of IL- 7 may be added to the agents of step b).
  • IL-7 is used in an amount comprised between 0.01 and 10 ng/ml, preferably between 0.1 and 1 ng/ml, even more preferably at about 0.5 ng/ml.
  • IL-7 can be obtained from a variety of sources. It may be purified or recombinant IL-7. IL-7 is commercially available from different companies, for example R&D Systems or PeproTech.
  • step b) is carried out for an amount of time t(b) sufficient to mature DC.
  • this amount of time t(b) is comprised between about 12 and about 72 hours, preferably between about 16 and about 48 hours, even more preferably for about 24 hours.
  • step b) is carried out for a shorter amount of time t(b) comprised between 0 and 12 hours.
  • the blood sample or PBMC sample subjected to the method of the invention contains a co-culture of DC at various maturation stages (monocytes, immature DC, mature DC) and of T cells, amongst other cells. It is believed that, within this heterogeneous population of cells, the DC will take up the Ag and present it to on their surface to the T cells, which will thereby be stimulated in an Ag-specif ⁇ c manner.
  • said Ag is a protein which can be obtained by recombinant DNA technology or by purification from different tissue or cell sources.
  • said protein has a length higher than 10 aminoacids, preferably higher than 15 aminoacids, even more preferably higher than 20 aminoacids with no theoretical upper limit.
  • Such proteins are not limited to natural ones, but also include modified proteins or chimeric constructs, obtained for example by changing selected aminoacid sequences or by fusing portions of different proteins.
  • said Ag is a synthetic peptide.
  • said synthetic peptide is 3-40 aminoacid-long, preferably 5-30 aminoacid-long, even more preferably 8-20 aminoacid-long.
  • Synthetic peptides can be obtained by Fmoc biochemical procedures, large-scale multipin peptide synthesis, recombinant DNA technology or other suitable procedures. Such peptides are not limited to natural ones, but also include modified peptides or chimeric peptides, obtained for example by changing selected aminoacid sequences or by fusing portions of different proteins.
  • the Ag is a crude or partially purified tissue or cell preparation obtained by different biochemical procedures (e.g., lysis, subcellular fractionation, density gradient separation) known to the expert in the art.
  • step said Ag should be introduced.
  • the Ag is a protein, it will generally be added during steps a).
  • the Ag is a peptide, it can be added at step b) rather than at step a).
  • step b there is no clear aminoacid length cutoff to predict whether a given peptide will be directly bound to MHC Class II molecules or will be taken up and processed by dendritic cells prior to presentation on MHC Class II molecules, it falls within the ability of the person skilled in the art to optimize the time of addition of intermediate length peptides for each case.
  • Enzyme-linked immunospot (ELISpot):
  • Non-adherent cells from pre-culture wells are transferred to a plate which has been coated with the desired anti-cytokine capture antibodies (Abs; e.g., anti-IFN- ⁇ , -IL- 10, -IL-2, -IL-4).
  • Abs anti-IL- ⁇ , -IL- 10, -IL-2, -IL-4.
  • Revelation is carried out with biotinylated secondary Abs and standard colorimetric detection methods such as streptavidin-alkaline phosphatase and NBT-BCIP and the spots counted.
  • ELISpot readouts are then expressed as spot- forming cells (SFCyiO 6 PBMCs.
  • Cytokines released in the culture supernatant are measured by different techniques, such as enzyme-linked immunosorbent assays (ELISA), BD cytometric bead array, Biorad Bio-Plex assay and others.
  • ELISA enzyme-linked immunosorbent assays
  • BD cytometric bead array Biorad Bio-Plex assay and others.
  • Ag-reactive T cells recognizing specific peptide epitopes are detected, using either commercially available reagents (e.g., Prolmmune MHC Class II Ultimers) or in-house generated ones (e.g., from Dr. G.T. Nepom, Benaroya Research Institute, Seattle, USA) [Novak et al, J.Clin.Invest. 104:R63, 1999].
  • commercially available reagents e.g., Prolmmune MHC Class II Ultimers
  • in-house generated ones e.g., from Dr. G.T. Nepom, Benaroya Research Institute, Seattle, USA
  • Upregulation of activation markers (e.g., CD69, CD25): With this procedure, Ag-specif ⁇ c T cell responses are detected by their differential expression of activation markers exposed on the membrane following Ag- recognition.
  • Cytokine capture assays This system developed by Miltenyi Biotech is a valid alternative to the ELISpot to visualize Ag-specif ⁇ c T cells according to their cytokine response. In addition, it allows the direct sorting and cloning of the T cells of interest (see below).
  • CD 154 assay This procedure has been recently described in detail [Chattopadhyay et al., Nat. Med. 11 :1113, 2005; Frentsch et al., Nat. Med. 11 : 1118, 2005]. It is limited to detection of Ag-specific CD4+ T cells.
  • CD 107 assay This procedure has been recently described in detail [Chattopadhyay et al., Nat. Med. 11 :1113, 2005; Frentsch et al., Nat. Med. 11 : 1118, 2005]. It is limited to detection of Ag-specific CD4+ T cells.
  • CD 107 assay This procedure has been recently described in detail [Chattopadhyay et al., Nat. Med. 11 :1113, 2005; Frentsch et al., Nat. Med. 11 : 1118, 2005]. It is limited to detection of Ag-specific CD4+ T cells.
  • CD 107 assay This procedure has been recently described in detail [Chattopadhyay
  • Unfractionated PBMCs or even unfractionated blood can be used, either fresh or frozen. There is no need for preliminary purification steps, making the technique simpler and less demanding in terms of blood volumes; 3. No preliminary long-term expansion is required;
  • the same stimulation technique can be used to expand and subsequently sort Ag- specific CD4+ T cells and to generate CD4+ T cell lines and clones for further characterization.
  • the invention therefore also relates to a method for diagnosing a disease in a subject comprising the following steps: a) culturing a blood sample or a PBMC sample obtained from said subject in a medium which induces the differentiation of DC; b) optionally, maturing said DC; c) detecting T cell responses; wherein one or more disease-associated Ags are added during steps a) and/or b).
  • the invention also relates to a method for monitoring the effects of an immune therapy in a subject suffering from a disease comprising the following steps: a) culturing a blood sample or a PBMC sample obtained from said subject in a medium which induces the differentiation of DC; b) optionally, maturing said DC; c) detecting T cell responses; wherein one or more disease-associated Ags are added during steps a) and/or b).
  • the method of the invention for stimulating Ag-specif ⁇ c T cell responses can be useful both for diagnosing a disease and for monitoring the immunological effects of an immune therapy in several settings.
  • said disease is selected from the group consisting of autoimmune diseases.
  • This group comprises, but is not limited to, type 1 diabetes (TlD), Wegener's granulomatosis, Crohn's disease, celiac disease and multiple sclerosis.
  • said disease is selected from the group consisting of cancer disease.
  • This group comprises, but is not limited to, melanoma, colon cancer, renal cancer and haematological malignancies such as leukemias, lymphomas and multiple myeloma.
  • said disease is selected from the group consisting of infectious diseases.
  • infectious diseases comprises, but is not limited to, diseases caused by infectious agents such as M. tuberculosis, HIV, hepatitis C virus, cytomegalovirus, Epstein-Barr virus, influenza viruses.
  • said disease is a graft-vs-host disease complicating bone marrow transplantation and similar procedures.
  • the method of the invention can be used to detect one or more Ag-specif ⁇ c T cell responses which are correlated with the disease, preferably an autoimmune disease.
  • the method can be used to detect preproinsulin- or glutamic acid decarboxylase (GAD)-specif ⁇ c T cell responses which are correlated with type 1 diabetes.
  • GID glutamic acid decarboxylase
  • monitoring immune therapy refers to measurement of changes in T cell responses induced in a given subject following in vivo administration of immune modulating agents. For monitoring applications, different types of situations are found, according to the type of disease.
  • immune modulatory therapies can be used to turn off pathological immune responses.
  • One strategy to accomplish this result relies on non- Ag-specific interventions based on a number of immune modulatory agents.
  • agents such as cyclosporin A (Stiller et al., Science 223:1362, 1984; Feutren et al., Lancet 19:119, 1986; Bougneres et al., Diabetes 39:1264, 1990), Daclizumab, mycophenolate mofetil, rapamycin, interleukin-2, anti-CD3 monoclonal antibodies (Herald et al., N.Engl.J.Med. 346:1692, 2002; Keymeulen et al., N.EnglJ.Med.
  • anti-CD20 monoclonal antibodies such as Rituximab, autologous non-myeloablative hematopoietic stem cell transplantation (Voltarelli et al., JAMA 297:1568, 2007), autologous cord blood cell infusion (Haller et al., Exp.Hematol. 36:710, 2008), vitamin D, T regulatory cell adaptive therapies have been, are being or are likely to be tested for TlD prevention and/or intervention.
  • a second approach relies on Ag-specif ⁇ c strategies, i.e., administration of a disease-related Ag in a tolerogenic form.
  • agents such as (pro)insulin (DPT-I, N.EnglJ.Med.
  • Immune responses against cancer or infected cells/infectious agents are therefore physiological adaptations trying to counter-act disease.
  • These physiological mechanisms can be therapeutically boosted, using either non-Ag- specific strategies (for example, cytotoxic T lymphocyte-associated antigen 4 blockade, alone or in combination with various agents, in melanoma; Yuan et al., Proc.Natl.Acad.Sci. USA 105:20410, 2008; Maker et al., Ann.Surg.Oncol. 12:1005, 2005) or Ag-specif ⁇ c approaches, i.e., administration (so called vaccination) of disease-related Ag(s) in an immunogenic form.
  • non-Ag-specific strategies for example, cytotoxic T lymphocyte-associated antigen 4 blockade, alone or in combination with various agents, in melanoma; Yuan et al., Proc.Natl.Acad.Sci. USA 105:20410, 2008; Maker et al., Ann
  • the method for stimulating Ag-specific T cell responses of the invention can therefore be very useful to monitor induction of these immune changes.
  • Step c) of detecting T cell responses can be performed as described above, for example by measuring the amount of a given cytokine which is secreted.
  • step c) of detecting T cell responses is performed by ELISpot.
  • disease-associated antigens refers to proteins or peptides which constitute the molecular targets of an immune response. Said molecular targets are expressed by the tissue(s) targeted by the immune response. Expression of disease-associated Ags can be limited to the target tissue or be extended to additional body compartments. Disease-associated Ags can be initially identified as being targets of autoantibody or T cell immune responses, or based on their selective expression by the target tissue.
  • disease-associated protein antigens are preproinsulin (PPI), glutamic acid decarboxylase (GAD), insulinoma-associated protein 2 (IA-2), islet-specific glucose-6-phosphatase catalytic-subunit-related protein (IGRP) and zinc transporter 8 (ZnT8) for TlD; myeloperoxydase and proteinase 3 for Wegener's graulomatosis; myelin oligodendrocyte glycoprotein (MOG) and myelin basic protein (MBP) in multiple sclerosis; gliadins in celiac disease; tyrosinase, melan-A, MART-I, gplOO and NY- ESO-I in melanoma cancer; ESAT-6 for M. tuberculosis infection; and gag for HIV infection.
  • PPI preproinsulin
  • GAD glutamic acid decarboxylase
  • IA-2 insulinoma-associated protein 2
  • IGRP islet-specific
  • Examples of disease-associated peptide Ags are derived from the above said protein Ags following processing by Ag-presenting cells - including DC - and presentation in the context of different HLA Class I or Class II molecules. Therefore, said peptide Ags are different depending not only on their source Ag, but also on the HLA molecules by which they are presented. For example, a list of T ID-associated peptide Ags for both mouse and human can be found in DiLorenzo et al., Clin. Exp. Immunol. 148:1, 2007.
  • the immune response mounted against disease-associated Ags can be a pathological one (i.e., in the case of autoimmune diseases) or a physiological, potentially beneficial one, aimed at limiting the consequences of another ongoing pathological process (i.e., in the case of cancer of infectious diseases).
  • pathological or physiological immune responses underlying said diseases detection of such responses can be used to diagnose these diseases, or to follow their natural or therapeutically modified evolution.
  • the method described herein can therefore be applied to both immune diagnosis and monitoring (e.g., immune staging, therapeutic follow-up) of said diseases.
  • therapeutic proteins refers to protein or peptide compounds of any aminoacid length which are administered or are planned to be administered in vivo to human subjects to achieve a therapeutic effect.
  • therapeutic proteins are, but are not limited to, disease-associated Ags (as defined above), antibodies of different species (either in their native form or partially/fully humanized), cytokines, hormones or hormone analogues, coagulation factors, enzymes, bacterial or viral proteins.
  • Such proteins are not limited to natural ones, but also include modified proteins or chimeric constructs, obtained for example by changing selected aminoacid sequences or by fusing portions of different proteins.
  • modified proteins or chimeric constructs obtained for example by changing selected aminoacid sequences or by fusing portions of different proteins.
  • immunogenicity of therapeutic proteins is of relevance.
  • One first therapeutic setting concerns the use of disease-associated Ags (as defined above) for in vivo administration, with the aim of inducing a tolerogenic effect (e.g., in the case of autoimmune diseases) or an immunogenic effect (e.g., in the case of cancer or infectious diseases). It is important to first evaluate in vitro the potential to achieve said desired therapeutic effect.
  • the aim is not to induce immunogenic responses of any kind to the administered protein, but rather to avoid such responses so to allow said protein to achieve the therapeutic effect for which it is designed.
  • Example of such settings include, without being limited to, cytokine-based immune therapies, hormone replacement therapies and replacement therapies for coagulation factors (e.g., Factor VIII in Haemophilia A) or enzymatic deficits (e.g., beta-glucuronidase in mucopolysaccharidosis VII).
  • cytokine-based immune therapies include, without being limited to, cytokine-based immune therapies, hormone replacement therapies and replacement therapies for coagulation factors (e.g., Factor VIII in Haemophilia A) or enzymatic deficits (e.g., beta-glucuronidase in mucopolysaccharidosis VII).
  • coagulation factors e.g., Factor VIII in Haemophilia A
  • enzymatic deficits e.g., beta-glucuronidase in
  • the invention therefore also relates to a method for evaluating the immunogenicity of a therapeutic protein comprising the following steps: a) culturing a blood sample or a PBMC sample in a medium which induces the differentiation of DC; b) optionally, maturing said DC; c) detecting T cell responses; wherein said therapeutic protein is added during steps a) and/or b).
  • epitope discovery also known as “epitope mapping”
  • epitope mapping i.e. for screening epitopes in order to select those eliciting an Ag-specif ⁇ c T cell response.
  • epitope refers to the portion of a protein Ag recognized by a T cell.
  • Epitopes are peptides of different aminoacid length which can bind to major histocompatibility complex (MHC) Class I or Class II molecules.
  • MHC major histocompatibility complex
  • TCR T cell receptor
  • epitopes are the defined molecular targets of T cells, it is often relevant to precisely identify such targets to design appropriate peptides to be used for in vitro applications (e.g., detection of Ag-specific T cell responses for diagnostic, prognostic or therapeutic purposes) or for in vivo administration (e.g., epitope-based tolerogenic therapies in autoimmune diseases; or epitope-based vaccinations in cancer and infectious diseases). Furthermore, definition of common rules governing epitope binding to a given MHC molecule (e.g.
  • HLA-A2, A*0201; or HLA-DR4, DR*0401) and/or triggering of TCR signalling and T cell activation is often pursued with the aim of developing computerized algorithms capable of predicting the behaviour of a given epitope. Developing of such algorithms frequently requires availability of large experimental data sets.
  • the invention therefore also relates to a method for screening candidate epitopes comprising the following steps: a) culturing a blood sample or a PBMC sample in a medium which induces the differentiation of DC; b) optionally, maturing said DC; c) detecting T cell responses; wherein a candidate epitope is added during steps a) and/or b).
  • a method for screening candidate epitopes comprising the following steps: a) culturing a blood sample or a PBMC sample in a medium which induces the differentiation of DC; b) optionally, maturing said DC; c) detecting T cell responses; wherein a candidate epitope is added during steps a) and/or b).
  • Yet another application of the method of the invention concerns its use for producing T cell clones.
  • the invention relates to a method for producing T cell clones displaying specific immunological properties from a subject comprising the following steps: a) culturing a blood sample or a PBMC sample obtained from said subject in a medium which induces the differentiation of DC; b) optionally, maturing said DC; c) isolating at least one T cell displaying said specific immunological properties; wherein an Ag is added during steps a) and/or b).
  • Said specific immunological properties include, but are not limited to, recognition by the isolated T cells of the Ag added during step a) and/or b).
  • said specific immunological properties may also include the production of IFN- ⁇ or the ability to exert cytotoxic effects on cells presenting the recognized Ag.
  • T cell clones producing IFN- ⁇ or displaying cytotoxicity can be useful for example for treatment of cancer and infectious diseases.
  • T cell clones producing IL-IO can be used as regulatory T cells for the treatment of autoimmune diseases.
  • T cells once isolated from a blood sample or a PBMC sample.
  • T cell cloning methods can be found in Reijonen et al, Diabetes 51 :1375, 2002; Mallone et al., Blood 106:2798, 2005; Mannering et al.,
  • the person skilled in the art is also familiar with methods suitable to isolate said Ag- specific T cells in a viable state based on different immunological properties. For example, selection of IFN- ⁇ - or IL-10-producing T cells may be obtained by Miltenyi cytokine capture assays. As another example, selection of cytotoxic T cells may be obtained based on upregulation of CD107 [Betts et al, J. Immunol. Methods 281 :65, 2003].
  • Example 1 PBMC-derived accelerated co-cultured DC (acDC) amplify the Ag- specific responses of co-cultured T cells - protein Ag stimulation
  • PBMCs 2.5xlO 6 cells/well were plated into 48-well plates in AIM- V medium (Invitrogen) supplemented with 1000 U/ml GM-CSF, 500 U/ml IL-4 (both from R&D Systems), and relevant protein Ags (10 ⁇ g/ml).
  • the protein antigens tested were tetanus toxoid (TTX), M. tubercolosis purified protein derivative (PPD), exavalent vaccine Infanrix hexa (Glaxo SmithKline), proinsulin (PI), glutamic acid decarboxylase (GAD), insulin C-peptide, pre-PI leader sequence, myeloperoxidase, proteinase 3.
  • TTX tetanus toxoid
  • PPD M. tubercolosis purified protein derivative
  • Infanrix hexa Gaxo SmithKline
  • proinsulin PI
  • GAD glutamic acid decarboxylase
  • Maturation protocol A 1000 U/ml TNF- ⁇ , 10 ng/ml IL- l ⁇ (both from R&D) and 1 ⁇ M PGE 2 (Calbiochem).
  • Maturation protocol B anti-CD40 monoclonal antibody (mAb; clone G28-5, 10 ⁇ g/ml), IFN- ⁇ 2a (Roferon-A, Roche; 1 ,000 U/ml).
  • This protocol was suitable to detect Ag-specif ⁇ c CD4+ T cell responses producing for example IFN- ⁇ and IL- 10.
  • Maturation protocol C LPS 100 ng/ml and low-dose IL-7 (0.5ng/ml). This protocol was suitable to detect Ag-specif ⁇ c CD4+ T cell responses producing for example IFN- ⁇ , but not IL-10, due to a large increase in basal (unstimulated) IL-10 secretion.
  • Maturation protocol D poly LC 20 ⁇ g/ml and low-dose IL-7 (0.5ng/ml). This protocol was suitable to detect Ag-specif ⁇ c CD4+ T cell responses producing for example IFN- ⁇ , but not IL-10, due to a large increase in basal (unstimulated) IL-10 secretion.
  • T cell detection procedure ELISpot. Non-adherent cells were washed, resuspended in fresh AIM-V medium and distributed in triplicate wells (0.3xl0 6 cells/well) of 96- well PVDF plates, which had been coated overnight with the desired anti-cytokine capture Abs (e.g., anti-IFN- ⁇ , -IL-10, -IL-2, -IL-4; all from U-CyTech). No further Ags or cytokines were added, and the plates were incubated for 6h at 37°C, 5% CO 2 . Revelation was carried out with biotinylated secondary Abs and standard colorimetric detection such as streptavidin- alkaline phosphatase and NBT-BCIP.
  • anti-cytokine capture Abs e.g., anti-IFN- ⁇ , -IL-10, -IL-2, -IL-4; all from U-CyTech.
  • Characterization of acDC revealed a phenotype identical to that of conventional 7 day DC.
  • CD14 down-regulation was paralleled by increased expression of HLA-DR and co-stimulatory molecules, while dextran uptake decreased upon maturation.
  • acDC were much more effective when matured with pro-inflammatory stimuli, as compared to acDC left immature (i.e., treated with GM-CSF and IL-4 only).
  • the efficiency of acDC was similar to that of conventional 7 day DC and was similarly effective at amplifying different cytokine responses, including IFN- ⁇ , IL-IO, IL-2 and IL-4.
  • the median increase in specific signal with acDC matured with TNF- ⁇ , PGE2 and IL-IB as compared to monocytes was 2.2-fold (range 1.5-8.7) for IFN- ⁇ and 1.4-fold (range 1.2-5.0) for IL-10.
  • Example 2 peptide Ag stimulation.
  • Example 1 The same experiment as described in Example 1 was carried out, using peptide antigen stimulation instead of protein Ag stimulation.
  • peptide Ags tested were: Influenza matrix protein (MP) 58 -66, Influenza hemagglutinin (HA) 30 6-3i8, GAD 5 55_567, GADii4_i23, PI BIO-IS.
  • Example of peptides used as negative controls were pyruvate dehydrogenase (PD) 5 _i 3 and collagen II (CII) 2 6 1-273 .
  • the acDC culture protocol can be used to stimulate T cells with either protein or peptide Ags.
  • Example 3 acDC induction directly in blood. Cytokine cocktails and protein/peptide Ags were added as in Example 1 (for protein Ags) or Example 2 (for peptide Ags) directly into freshly drawn heparinized blood samples, without any preliminary PBMC purification or blood dilution. At the end of the 48h culture, plasma and/or PBMCs were recovered and analyzed for Ag-specif ⁇ c T cell responses by plasma cytokine measurement using ELISA (R&D), cytometric bead array (BD), or Bio-Plex (Biorad) assays, or by Miltenyi cytokine capture assays on the cellular fraction following red blood cell lysis.
  • ELISA ELISA
  • BD cytometric bead array
  • Biorad Bio-Plex
  • Example 4 T cell expansion, sorting and cloning downstream of the acDC culture and T cell response analysis.
  • the acDC culture system is also suitable: 1) for sorting Ag-specif ⁇ c T cells for further functional characterization (e.g., by RT-PCR techniques); and 2) for generating T cell lines and clones for further analyses.
  • PBMCs were preliminarily labeled with CFSE (carboxyfluorescein succinimidyl ester, 0.5-1 ⁇ M), subjected to the acDC culture and transferred to ELISpot plates as above. After the 6 h ELISpot incubation, cells were recovered and put back into culture for an additional 5 days without further stimuli. At the end of this culture, proliferating CFSE low cells were single-cell sorted and further expanded through 3 rounds of stimulation using previously described protocols [Mannering et al, J. Immunol. Methods 298:83, 2005].
  • CFSE carboxyfluorescein succinimidyl ester
  • TTX-specific response was detected by ELISpot with a frequency of 0.044%, which correlated with selection of a TTX-specif ⁇ c CFSE low fraction of 3.0%, corresponding to an expansion of ⁇ 68-fold.
  • the acDC-based culture proved superior to conventional monocyte-based expansions, which yielded 10-fold less TTX-specific cells (0.29%).
  • the TTX-specific CFSE low fraction was sorted and cloned, yielding TTX-specif ⁇ c CD4 + clones. This approach also confirmed that the responses detected from acDC cultures are indeed Ag-specif ⁇ c.
  • Example 5 the acDC culture system detects ⁇ -cell-specific CD4 + T-cell responses.
  • Type-1 diabetes is a T-cell-mediated autoimmune disease targeting insulin- producing ⁇ -cells. Its incidence is steadily increasing (up to 15 new diagnoses/100, 000/year in France; 3-4% increase in incidence per year). Given its distinctive epidemiology (it mainly affects children and young adults life-long), it is a chronic, costly and invalidating disease, leading to severe complications: cardiovascular disease, nephropathy and end-stage renal disease, retinopathy and blindness.
  • TlD clinical onset and diagnosis is a late event in the pathogenic cascade, which takes place when the majority of the ⁇ -cells have already been destroyed over the previous months/years by autoreactive T cells. At that stage, little space is left for cause-related therapies aimed at correcting immune mechanisms, and insulin replacement is the only therapeutic option. There is therefore an important lack of suitable biomarkers for disease prediction and follow-up, and of appropriate antigen (Ag)-specif ⁇ c therapies capable of selectively turning off ⁇ -cell-specific T cell responses while avoiding generalized immune suppression.
  • Ag antigen
  • TlD For TlD, autoantibodies currently in use have important limitations, as 15% of TlD patients are autoantibody-negative; autoantibodies do not predict time to TlD onset; and they do not change following successful immune interventions.
  • T cell responses whether CD 8+ T cell responses or CD4+ T cell responses, can be used as early biomarkers. Cytokine ELISpot is a widely used assay for the investigation of specific immune responses in various conditions. CD8+ T-cell responses can be readily detected by this method.
  • TlD adults drawn at diagnosis were characterized by a high prevalence (83.3%) of proinsulin (P ⁇ )-specif ⁇ c responses, which contrasted with the rarity of these responses in longstanding patients (5.4%; P ⁇ 0.0001).
  • P ⁇ proinsulin
  • GAD-specif ⁇ c responses were similarly represented - although at lower frequencies - irrespective of TlD duration.
  • new-onset TlD children did not display any Pi-specific T cell response. Healthy controls (both adults and children) did not display any Pi-specific T cell response except in two cases (frequency 8.7%).

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EP2419730A1 (en) 2012-02-22
US9091679B2 (en) 2015-07-28
EP2419730B1 (en) 2016-08-10
US20120094865A1 (en) 2012-04-19
JP5776684B2 (ja) 2015-09-09

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